Extended spectrum beta-lactamases A. Beta-lactam antibiotics a. Structure b. Types c. Action d. Mechanism of resistances B. Beta-lactamases a. Classical beta-lactamases b. Extended spectrum beta-lactamases (ESBL) c. Non-TEM, non-SBV ESBL d. Inhibitor Resistant TEM (IRT) C. Definition, classification and properties of ESBL D. Epidemiology and risk factors E. Laboratory detection and identification of ESBLs a. Screening, phenotypic and genotypic methods b. Co-production of ESBL and AmpC beta-lactamases F. Beta-lactamase inhibitors G. Multiple drug resistance H. Treatment options against ESBL producers A. Beta-lactam antibiotics A β-lactam (beta-lactam) ring is a four-membered cyclic amide consisting of three carbon atoms and one nitrogen atom. It is named so, because the nitrogen atom is attached to the β-carbon relative to the carbonyl (C=O). Antibiotics possessing this structure are called beta-lactam antibiotics. Penams contain a β-lactam ring fused to a 5- membered ring, where one of the atoms in the ring is a sulfur and the ring is fully saturated. A carbapenam is a β-lactam compound that is a saturated carbapenem. They exist primarily as biosynthetic intermediates on the way to the carbapenem antibiotics. A clavam is a molecule similar to a penam, but with an oxygen atom substituted for the sulfur. Thus, they are also known as oxapenams. Carbapenems are very similar to the penams, but the sulfur atom of the unsaturated structure is replaced with a carbon atom. Penem is a type of unsaturated β-lactam, which is similar in structure to carbapenems but penems have a sulfur atom instead of carbon. Carbacephems are similar to cephems but with a carbon substituted for the sulfur. An oxacephem is a molecule similar to a cephem, but with oxygen substituted for the sulfur. Monobactams are β-lactam compounds wherein the β-lactam ring is alone and not fused to another ring. β-lactams are classified according to their core ring structures ¾ β-lactams fused to saturated five-membered rings: β-lactams containing thiazolidine rings are named penams (penicillins) β-Lactams containing pyrrolidine rings are named carbapenams (intermediates) β-lactams fused to oxazolidine rings are named oxapenams or clavams (clavulanic acid) ¾ β-lactams fused to unsaturated five-membered rings: β-lactams containing 2,3-dihydro-1H-pyrrole rings are named carbapenems (imipenem) β-lactams containing 2,3-dihydrothiazole rings are named penems (faropemem) ¾ β-lactams fused to unsaturated six-membered rings: β-lactams containing 3,6-dihydro-2H-1,3-thiazine rings are named cephems (cephalosporins) β-lactams containing 1,2,3,4-tetrahydropyridine rings are named carbacephems (loracarbef) β-lactams containing 3,6-dihydro-2H-1,3-oxazine rings are named oxacephems (moxalactam) ¾ β-lactams not fused to any other ring are named monobactams (aztreonam) Penicillin was inadvertently discovered by Alexander Fleming in 1928 when he observed that contaminating mould, Penicillium notatum inhibited growth of Staphylococcus on a culture plate. It took 12 years for the compound to be purified and used in treatment. It was due to the combined efforts of Ernst Boris Chain, Edward Abraham, Howard Florey and Norman Heatley that penicillin was obtained in pure and usable form. a. Structure: The molecular formula of penicillin is R-C9H11N2O4S, where R is the variable side chain. 6-amino penicillanic acid is the core of penicillins and is used as the main starting block for the preparation of numerous semisynthetic penicillins. The basic structure of penicillins consists of a thiazolidine ring connected to a β-lactam ring with an attached side chain (R). The R side chain determines many of the antibacterial and pharmacological characteristics of the penicillin type. Penicillin is available in various forms including benzylpenicillin (penicillin G), procaine benzylpenicillin, benzathine benzylpenicillin and phenoxymethylpenicillin (penicillin V). Only Penicillin V is administered orally. In order to overcome resistance to penicillin by the penicillinase enzyme, ampicillin was introduced in 1950s. The first semi- synthetic penicillin introduced in the clinical practice was phenethicillin. This was followed by ampicillin, amoxycillin, the β-lactamase-resistant isoxazolyl penicillins (methicillin, oxacillin, cloxacillin, dicloxacillin and flucloxacillin), and antipseudomonal penicillins (carbenicillin, piperacillin and ticarcillin). The active part of penicillins and related antibiotics (cephalosporins, monobactam and carbapenem) is the beta-lactam ring. Cephalosporin was first isolated in 1948 from a fungus Cephalosprium (now Acremonium) sps by Italian scientist Giuseppe Brotzu. Cephalosporins are basically semi-synthetic derivatives of the cephalosporin C, which was isolated from the fungus. The first commercial preparation- cefalotin (cephalothin) was launched in 1964. The nucleus of cephalosporin antibiotics is 7-aminocephalosporanic acid © Sridhar Rao P.N (www.microrao.com) 2 (a β–lactam ring). It is made of 4-membered β-lactam ring and 6-membered dihydrothiazolidine ring with two side chains R1 and R2. Modifications in the side chain have resulted in several types of cephalosporins, which are grouped into several generations. Cephalosporins are more effective against gram negative bacteria. The cephamycins are similar to the cephalosporins, but have a methoxy group at position 7 of the beta-lactam ring of the 7-aminocephalosporanic acid nucleus. Mechanism of action is similar to those of penicillins. First generation cephalosporins were introduced in the 1960s. In the late 1970s and early 1980s, penicillinase resistant oxyimino- aminothiazolyl (3rd generation) cephalosporins were introduced. The initial cephalosporins were designated first-generation cephalosporins whereas, later cephalosporins, that had more extended-spectrum of activity were classified as second-generation. Each newer generation of cephalosporins has significantly greater Gram-negative antimicrobial properties than the preceding generation. Fourth-generation cephalosporins have true broad-spectrum activity. There are disagreements over “generations” and the placements of antibiotics in these generations. b. Types of Beta-lactam antibiotics: Penicillins Narrow β-lactamase Benzylpenicillin, Benzathine spectrum susceptible benzylpenicillin, Procaine benzylpenicillin,Phenoxymethylpenicillin β-lactamase resistant Cloxacillin (Dicloxacillin, Flucloxacillin), Oxacillin, Meticillin, Nafcillin Broad Aminopenicillins Amoxicillin, Ampicillin, Epicillin spectrum Carboxypenicillins Carbenicillin, Ticarcillin, Temocillin Ureidopenicillins Azlocillin, Piperacillin, Mezlocillin Other Mecillinam, Sulbenicillin Cephalosporins 1st Cefazolin, Cefadroxil, Cefalexin, Cefaloridine, Cefalotin, generation Cefapirin, , Cefatrizine, Cefazedone, Cefazaflur, Cefradine, Cefroxadine, Ceftezole 2nd Cefaclor, Cefamandole, Cefminox, Cefonicid, Ceforanide, generation Cefotiam, Cefprozil, Cefbuperazone, Cefuroxime, Cefuzonam 3rd Cefixime, Ceftriaxone, Cefoperazone, Cefotaxime, generation Cefpodoxime, Cefdinir, Cefsulodin, Cefcapene, Cefdaloxime, Cefditoren, Cefetamet, Cefmenoxime, Cefodizime, Cefpimizole, Cefpiramide, Cefteram, Ceftibuten, Ceftiolene, Ceftizoxime 4th Cefepime, Cefpirome, Cefquinome, Cefozopran generation 5th Ceftobiprole, Ceftaroline fosamil generation Cephamycin Cefoxitin, Cefotetan, Cefmetazole Carbacephem Loracarbef Monobactam Aztreonam, Tigemonam, Carumonam, Tabtoxin Carbapenem Imipenem, Meropenem, Ertapenem, Doripenem, Biapenem, Panipenem © Sridhar Rao P.N (www.microrao.com) 3 c. Action of beta-lactam antibiotics: Bacterial cell wall is made up of glycopeptide called peptidoglycan (murein). The backbone of peptidoglycan is made of alternating units of N- acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). NAM has a tetrapeptide side chain attached to it. The tetrapeptide chain of gram negative bacteria consists of L-lysine, D-glutamic acid, diaminopimelic acid and d-alanine (d-alanine- d-alanine). The side chain of one backbone is cross linked to the side chain of the other backbone by pentaglycine cross-bridges in gram positive bacteria. The diaminopimelic acid in the side chain of one backbone is connected to d-alanine in the side chain of another backbone in gram negative cells. This cross-linking is brought about by a bacterial enzyme present in the periplasmic space called d-d transpeptidase. As the bacteria grow and multiply, they constantly remodel their cell walls by breaking down and building portions of cell wall. The β-lactam ring of the antibiotic is sterically identical to d-alanine-d- alanine. In the presence of β-lactam antibiotic in the periplasmic space, the transpeptidase enzyme mistakenly binds to the β-lactam antibiotic instead of its original substrate. Since the β-lactam antibiotic binds to the transpeptidase enzyme, the enzyme is also known as penicillin-binding-protein (PBP). The binding results in acylation of the enzyme leading to the production of an inactive penicilloyl-enzyme. As a result, further cross-linkages between the layers of peptidoglycan do not occur, which weakens the cell wall. Accumulation of cell-wall precursors are thought to initiate autolysis. The cell finally undergoes osmotic instability and lyses. Bacteria have multiple PBPs. E. coli has seven PBPs, each with a distinct role. PBP 1A and B are important in cell elongation, PBP 2 maintain rod shape of cell wall, PBP 3 forms septum between dividing cells. PBPs 4, 5, and 6 are thought to be non-essential. The sensitivity of